Vaccine

29 (2011) 8222–

8229

Contents

lists

available

at

SciVerse

ScienceDirect

Vaccine

j

o u r n a

l

h o

m e

p a g

e :

w w w . e l s e v i e r . c o m / l o c a t e / v a c c i n e

Mucosal

immunization

with

Shigella

flexneri

outer

membrane

vesicles

induced

protection

in

mice

A.I.

Camacho

a

,

J.

de

Souza

a

,

S.

Sánchez-Gómez

a

,

M.

Pardo-Ros

a

,

J.M.

Irache

b

,

C.

Gamazo

a

,

∗

a

Department

of

Microbiology,

University

of

Navarra,

31008

Pamplona,

Spain

b

Department

of

Pharmacy

and

Pharmaceutical

Technology,

University

of

Navarra,

31008

Pamplona,

Spain

a

r

t

i

c

l

e

i

n

f

o

Article

history:

Received

7

June

2011

Received

in

revised

form

25

August

2011

Accepted

30

August

2011

Available online 10 September 2011

Keywords:
Shigella
Outer

membrane

vesicles

Vaccine
Nanoparticles
Adjuvant

a

b

s

t

r

a

c

t

Vaccination

appears

to

be

the

only

rational

prophylactic

approach

to

control

shigellosis.

Unfortunately,

there

is

still

no

safe

and

efficacious

vaccine

available.

We

investigated

the

protection

conferred

by

a

new

vaccine

containing

outer

membrane

vesicles

(OMVs)

from

Shigella

flexneri

with

an

adjuvant

based

on

nanoparticles

in

an

experimental

model

of

shigellosis

in

mice.

OMVs

were

encapsulated

in

poly(anhydride)

nanoparticles

prepared

by

a

solvent

displacement

method

with

the

copolymer

PMV/MA.

OMVs

loaded

into

NPs

(NP-OMVs)

were

homogeneous

and

spherical

in

shape,

with

a

size

of

197

nm

(PdI

=

0.06).

BALB/c

mice

(females,

9-week-old,

20

±

1

g)

were

immunized

by

intradermal,

nasal,

ocular

(20

␮g)

or

oral

route

(100

␮g)

with

free

or

encapsulated

OMV.

Thirty-five

days

after

administration,

mice

were

infected

intranasally

with

a

lethal

dose

of

S.

flexneri

(1

× 10

7

CFU).

The

new

vaccine

was

able

to

protect

fully

against

infection

when

it

was

administered

via

mucosa.

By

intradermal

route

the

NP-OMVs

formulation

increased

the

protection

from

20%,

obtained

with

free

extract,

to

100%.

Interestingly,

both

OMVs

and

OMV-NP

induced

full

protection

when

administered

by

the

nasal

and

conjuntival

route.

A

strong

association

between

the

ratio

of

IL-12p40/IL-10

and

protection

was

found.

Moreover,

low

levels

of

IFN-

␥

correlate

with

protection.

Under

the

experimental

conditions

used,

the

adjuvant

did

not

induce

any

adverse

effects.

These

results

place

OMVs

among

promising

candidates

to

be

used

for

vaccination

against

Shigellosis.

© 2011 Elsevier Ltd. All rights reserved.

1.

Introduction

According

to

World

Health

Organization

(WHO),

approximately

2.5

billion

cases

of

diarrhea

occurred

worldwide

which

results

in

1.5

million

deaths

among

children

under

the

age

of

five.

It

is

a

common

cause

of

death

in

developing

countries

and

the

second

most

com-

mon

cause

of

infant

deaths.

Among

the

main

causes,

Shigellosis

is

responsible

of

more

than

165

million

cases

annually,

leading

to

1.2

million

deaths

[1]

.

Furthermore,

many

cases

progress

into

serious

damages

in

their

intestinal

epithelium

that

will

limit

the

correct

nutrient

absorption

with

the

subsequent

sequel

for

life.

Shigella

spread

massively

within

the

community

and

from

person

to

per-

son,

and

hence,

prevention

relies

on

basic

sanitary

measures,

which

unfortunately

may

be

not

possible

applied

for

many

countries.

In

addition,

the

increasing

problem

of

antibiotic

resistance

alerts

on

the

urgent

need

of

protective

vaccines.

In

fact,

the

World

Health

Organization

has

made

the

development

of

a

safe

and

effective

vaccine

against

Shigella

a

high

priority

[1]

.

∗ Corresponding

author.

Tel.:

+34

9

48

42

56

88;

fax:

+34

9

48

42

56

49.

E-mail

address:

[e-mail]

(C.

Gamazo).

The

efforts

have

been

mainly

focussed

on

live

oral

vaccines

with

several

vaccine

candidates

on

clinical

trials

[2]

.

However,

develop-

ment

of

such

safe

Shigella

vaccine

is

being

problematical,

and

no

vaccine

is

still

available

[3]

.

Currently,

most

vaccines

in

development

are

acellular

vaccines

which

[2,4]

in

comparison

to

live-attenuated

or

whole

inactivated

organism,

are

safer.

However,

these

prototypes

require

adjuvants

to

achieve

a

more

effective

immune

response.

The

challenge

is

the

designing

of

formulations

able

to

enhance

the

immunogenicity

of

associated

antigens,

through

the

right

activation

of

the

immune

system,

and

susceptible

to

be

administered

by

mucosal

routes.

Previous

studies

of

our

group

have

evaluated

the

adjuvant

capa-

bility

of

nanoparticles

made

from

the

copolymer

of

methyl

vinyl

ether

and

maleic

anhydride

(Gantrez

AN

®

).

These

nanoparticles

demonstrated

their

ability

to

initiate

a

strong

and

balanced

mucosal

immune

response

and

then,

to

efficiently

induce

Th-1

subset

[5]

.

In

addition,

these

nanoparticles

loaded

with

different

antigens

have

showed

to

be

effective

against

experimental

challenges

with

Salmonella

or

Brucella

[6–9]

.

In

this

work

we

propose

the

use

of

outer

membrane

vesicles

(OMVs)

from

Shigella

as

the

source

of

relevant

antigens

to

be

included

in

the

acellular

vaccine.

OMVs

are

secreted

from

the

outer

membrane

of

a

large

variety

of

Gram-negative

bacteria,

during

0264-410X/$

–

see

front

matter ©

2011 Elsevier Ltd. All rights reserved.

doi:

10.1016/j.vaccine.2011.08.121

A.I.

Camacho

et

al.

/

Vaccine

29 (2011) 8222–

8229

8223

in

vitro

culture

and

during

infection

[10]

.

Currently,

there

have

been

described

many

functions

for

this

blebbing

process.

Functions

proposed

vary

from

facilitating

the

intracellular

bacterial

growth

within

phagocytes

[11]

,

to

the

delivery

of

effectors

molecules

critical

for

pathogen

dissemination

such

as

pathogen-associated

molecular

patterns

(PAMPs)

and

other

virulence

factors

to

host

cells

[12–14]

.

We

therefore

describe

here

the

preparation,

characterization

and

evaluation

of

Shigella

flexneri

outer

membrane

vesicles

in

order

to

be

used

in

vaccination.

We

obtained

the

OMVs

from

S.

flexneri

2a,

being

this

the

most

common

cause

of

shigellosis.

In

fact,

it’s

responsible

for

25–50%

of

all

cases

in

the

developing

world

[2]

.

The

protective

efficacy

of

OMVs

either

in

their

free

form

or

adju-

vanted

in

NP

were

tested

in

the

murine

pneumonia

model

[15]

after

immunization

with

one

single

dose

by

intradermal

or

mucosal

routes.

The

OMVs

formulations

obtained

and

characterized

here

were

found

to

induce

protection

in

mice

after

one

single

dose

against

a

lethal

dose

of

S.

flexneri

2a.

2.

Materials

and

methods

2.1.

Preparation

and

characterization

of

outer

membrane

vesicles

OMVs

were

obtained

from

S.

flexneri

2a

(clinical

isolate

from

Hospital

de

Navarra,

Pamplona,

Spain).

Vesicles

were

purified

from

a

method

adapted

from

Horstman

and

Kuehn

[16]

.

Bacteria

were

grown

in

LB

broth

overnight

to

early

stationary

phase.

Then,

bac-

teria

were

inactivated

with

a

solution

of

binary

ethylenimine

and

formaldehyde

(6

mM

BEI-0,

06%

FA,

6

h,

37

◦

C).

BEI

was

prepared

as

a

0.1

M

solution

by

cyclization

of

0.1

M

2-bromoethylamine

hydrobromide

(Sigma)

in

0.175

M

NaOH

solution

for

1

h

following

the

method

of

Bahnemann

[17]

.

Cells

were

removed

by

pelleting

(10,000

×

g,

10

min).

Supernatant

was

filtered

through

a

0.45

␮m

Durapore

PVDF

filter

(Millipore)

and

purified

by

ultradiafiltration

via

a

300-kDa

tangential

filtration

concentration

unit

(Millipore).

The

retentate

was

freezed

in

order

to

induce

larger

blebs

formed

through

reassociation

of

the

smaller

ones

into

multimicelles,

as

had

been

proposed

previously

[18]

.

Final

product

was

recovered

by

centrifugation

at

40,000

×

g,

2

h.

Total

protein

content

was

deter-

mined

by

the

method

of

Lowry,

with

bovine

serum

albumin

as

standard.

Lypopolysaccharide

(LPS)

content

was

determined

by

Purpald

assay

[19,20]

.

Briefly,

to

50

␮L

of

LPS

samples

or

stan-

dards

[21]

in

each

of

the

duplicate

wells

in

a

96-well

tissue

culture

plate,

50

␮L

of

32

mM

NaIO

4

was

added,

and

the

plate

was

incu-

bated

for

25

min

followed

by

addition

of

50

␮L

of

136

mM

purpald

reagent

in

2

N

NaOH.

After

further

incubation

for

20

min,

50

␮L

of

64

mM

NaIO

4

was

added,

and

the

plate

was

incubated

for

another

20

min.

The

foam

in

each

well

can

be

eliminated

by

addition

of

20

␮L

2-propanol.

The

absorbance

of

each

well

was

measured

by

a

plate

reader

at

550

nm.

Finally,

extract

was

resuspended

in

sample

buffer

1

×

and

analyzed

by

SDS-PAGE

and

immunoblotting,

using

polyclonal

pool

sera

from

patient

infected

with

S.

flexneri

(Clínica

Universidad

de

Navarra)

or

anti

IpaC

mAb

(kindly

provided

by

A.

Phalipon,

Institut

Pasteur).

The

morphology

of

the

vesicles

was

examined

by

Field

Emission

Scanning

Electron

Microscope.

2.1.1.

Outer

membrane

proteins

(OMPs)

Outer

membrane

proteins

(OMPs)

from

S.

flexneri

were

prepared

by

sequential

detergent

extraction

of

cell

envelopes

[18]

.

Briefly,

after

the

disruption

of

cells

by

sonication

(4

pulses

×

5

min,

power

2,

Branson

Sonifier

450),

whole

bacteria

were

removed

by

cen-

trifugation

at

6000

×

g,

15

min.

Cell

envelopes

were

recovered

from

supernatant

by

centrifugation

(40,000

× g,

1

h).

Pellet

was

resus-

pended

in

1%

Sarkosyl

(N-Lauryl

sarcosine,

Sigma

Chemical

Co.,

St.

Louis,

USA),

incubated

for

30

min

and

further

centrifuged

at

40,000

×

g,

1

h,

twice.

The

enriched

sediment

in

outer

membrane

proteins

was

suspended

in

0.5

M

Tris–HCl

(pH

6.8)

with

10%

SDS

(Lauryl

sulfate,

Sigma)

and

boiled

for

15

min

and

finally,

centrifuged

(20,000

×

g;

30

min).

The

OMPs

of

S.

flexneri

were

present

in

the

final

supernatant.

2.1.2.

Ipa

(invasion

plasmid

antigens)

proteins

secretion

assay

Secretion

of

Ipa

proteins

through

the

TTSS

(Type

three

secre-

tion

system)

was

induced

using

a

Congo

Red

secretion

assay

[22]

.

Exponential-phase

bacteria

were

harvested,

resuspended

in

10

␮M

Congo

Red/PBS,

and

incubated

at

37

◦

C

for

30

min.

Following

incu-

bation,

bacteria

were

pelleted

by

centrifugation,

and

supernatants

were

collected

and

passed

through

a

0.22

␮m-pore

filter.

Proteins

in

the

supernatants,

which

represent

proteins

secreted

through

the

TTSS,

were

then

concentrated

by

tricholoroacetic

acid

precip-

itation.

Finally,

extract

was

resuspended

in

sample

buffer

1

×

and

analyzed

by

SDS-PAGE

and

immunoblotting

using

anti-IpaB

or

-

IpaC

mAb

(kindly

provided

by

A.

Phalipon,

Institut

Pasteur).

2.2.

Preparation

and

characterization

of

nanoparticles

Poly(anhydride)

nanoparticles

were

prepared

by

a

modification

of

the

solvent

displacement

method

[6,23]

.

Briefly,

100

mg

of

the

copolymer

of

methyl

vinyl

ether

and

maleic

anhydride

(PVM/MA)

(Gantrez

®

AN

119;

M.W.

200

kDa)

was

dissolved

in

5

mL

acetone

under

magnetic

stirring

at

room

temperature.

On

the

other

hand,

5

mg

OMVs

were

dispersed

by

ultrasonication

with

a

probe

Micro-

son

TM

(Misonix

Inc.,

New

York,

USA)

in

10

mL

water

for

1

min.

After

dispersion,

nanoparticles

were

formed

by

addition

of

this

water

phase

containing

OMVs.

The

agitation

was

maintained

dur-

ing

15

min

in

order

to

allow

the

stabilization

of

the

system.

Organic

solvents

were

removed

under

reduced

pressure

(Büchi

R-144,

Switzerland).

The

obtained

nanoparticles

were

collected

by

cen-

trifugation

(27,000

×

g,

20

min,

4

◦

C)

and

washed

with

water

twice.

Finally,

particles

were

freeze-dried

using

sucrose

5%

as

crioprotec-

tor.

The

preparation

of

empty

nanoparticles

was

performed

in

the

same

way

in

the

absence

of

OMVs.

2.2.1.

Characterization

of

nanoparticles

The

particle

size

and

the

zeta

potential

of

nanoparticles

were

determined

by

photon

correlation

spectroscopy

(PCS)

and

electrophoretic

laser

Doppler

anemometry,

respectively,

using

a

Zetamaster

analyzer

system

(Malvern

Instruments

Ltd.,

Worces-

tershire,

UK).

The

diameter

of

the

nanoparticles

was

determined

after

dispersion

in

ultrapure

water

(1/10)

and

measured

at

25

◦

C

by

dynamic

light

scattering

angle

of

90

◦

C.

The

zeta

potential

was

determined

as

follows:

200

␮L

of

the

samples

was

diluted

in

2

mL

of

a

0.1

mM

KCl

solution

adjusted

to

pH

7.4.

The

morphology

of

the

vesicles

was

examined

by

Field

Emission

Scanning

Elec-

tron

Microscope

(Carl

Zeiss,

model

Ultra

Plus).

For

this

purpose

freeze-dried

formulations

were

resuspended

in

ultrapure

water

and

centrifuged

at

27,000

× g

for

20

min

at

4

◦

C.

Then,

supernatants

were

rejected

and

the

obtained

pellets

were

mounted

on

TEM

grids.

The

yield

of

the

nanoparticles

preparation

process

was

determined

by

gravimetry

as

described

previously

[23]

.

Briefly,

poly(anhydride)

nanoparticles,

freshly

prepared,

were

freeze-dried.

Then,

the

yield

was

calculated

as

the

difference

between

the

initial

amount

of

the

polymer

used

to

prepare

nanoparticles

and

the

weight

of

the

freeze-

dried

carriers.

2.2.2.

Loading

capacity

of

nanoparticles

The

yield

of

nanoparticles

was

calculated

from

the

difference

between

the

initial

amount

of

the

polymer

used

to

prepare

the

8224

A.I.

Camacho

et

al.

/

Vaccine

29 (2011) 8222–

8229

particles

and

the

weight

of

the

freeze-dried

samples.

The

abil-

ity

of

PVM/MA

nanoparticles

to

entrap

the

complex

antigen

was

directly

determined

after

degradation

of

loaded

nanoparticles

with

NaOH.

Briefly,

OMVs-loaded

Gantrez

nanoparticles

(15

mg)

were

dispersed

in

water

vortexing

1

min.

After

centrifugation

(27,000

×

g,

15

min)

pellet

was

resupended

in

NaOH

0.1

M

soni-

cated

(Microson

TM

Ultrasonic

cell

disruptor)

and

incubated

for

1

h

to

assess

the

total

delivery

of

the

associated

antigen.

After

this

time,

the

amount

of

antigen

released

from

the

nanoparticles

was

determined

using

microbicin

choninic

acid

(microBCA)

protein

assay

(Pierce,

Rockford,

CA,

USA).

In

order

to

avoid

interferences

of

the

process,

calibration

curves

were

made

with

degraded

blank

nanoparticles,

and

all

measurements

were

performed

in

triplicate.

2.2.3.

Determination

of

the

structural

integrity

and

antigenity

of

OMVs

Western

blot

analysis

was

used

as

a

qualitative

tool

to

exam-

ine

the

structure

of

the

antigens,

complementing

the

quantification

performed

by

microBCA.

To

accomplish

this

analysis,

the

protocol

for

nanoparticle

degradation

was

modified

in

order

to

avoid

any

interference

of

the

enzyme.

In

this

case,

after

nanoparticle

isolation,

15

mg

of

loaded

nanoparticles

were

dispersed

in

water

vortexing

1

min.

After

centrifugation

(27,000

×

g,

15

min)

pellet

was

resu-

pended

in

2

mL

of

a

mixed

of

dimethilformamide:acetone

(1:3)

(

−80

◦

C,

1

h).

After

centrifugation,

pellet

was

resuspended

in

ace-

tone

(

−80

◦

C,

30

min).

Finally,

extract

was

resuspended

in

sample

buffer

1

×

and

analyzed

by

SDS-PAGE

and

immunoblotting

using

polyclonal

sera

from

hyperimmunized

rabbit

with

S.

flexneri

[24]

.

2.3.

SDS-PAGE

and

immunoblotting

SDS-PAGE

was

performed

in

12%

acrylamide

slabs

(Criterion

XT,

Bio

Rad

Laboratories,

CA)

with

the

discontinuous

buffer

sys-

tem

of

Laemmli

and

gels

stained

with

Coomassie

blue

or

silver

staining.

After

electrophoresis,

gels

were

electroblotted

to

a

PVDF

(polyvinylidene

fluoride)

membrane

at

0.8

mA/cm

2

for

30

min.

Then,

membranes

were

soaked

overnight

in

a

blocking

solution

containing

3%

(w/v)

of

non-fat

milk

and

then

incubated

in

the

pres-

ence

of

different

sera,

described

above.

After

the

incubation,

the

membranes

were

washed

five

times;

the

anti-rabbit

or

human

Ig-

alkaline

phosphatase

conjugate

was

added,

followed

by

incubation

for

an

additional

hour.

The

membranes

were

exhaustively

washed

and

the

antibody–antigen

complexes

were

visualized

after

addition

of

the

substrate/chromogen

solution

(H

2

O

2

/cloronaftol).

2.4.

Active

immunization

and

challenge

All

mice

were

treated

in

accordance

with

institutional

guide-

lines

for

treatment

of

animals

(Protocol

087/06

of

animal

treatment,

approved

in

1

October

2007

by

the

Ethical

Comity

for

the

Animal

Experimentation,

CEEA,

of

the

University

of

Navarra).

Nine-week-

old

BALB/c

mice

(20

±

1

g)

were

separated

in

randomized

groups

of

6

animals

and

immunized

with

OMVs

either

free

or

encapsulated

in

PVM/MA

NPs

by

intradermal,

nasal,

ocular

(20

␮g

of

extract)

or

oral

route

(100

␮g

of

extract).

The

scheme

of

administration

and

doses

are

summarized

in

Table

1

.

Challenge

infection

was

performed

on

day

35

intranasally

with

a

lethal

dose

of

1

×

10

7

UFC/Mouse

of

S.

flexneri

2a

(clinical

isolate)

grown

to

logarithmic

phase

and

suspended

in

20

␮L

of

prewarmed

PBS.

The

number

of

dead

mice

after

challenge

was

recorded

daily.

2.5.

Measurement

of

immune

response

in

the

mouse

Blood

samples

were

collected

from

the

reto-orbital

plexures

of

anesthetized

mice.

2.5.1.

ELISA

The

antibody

response

was

measured

by

an

enzyme-linked

immunosorbent

assay

(ELISA).

In

brief,

96-well

microtiter

plates

(MaxiSorb;

Nunc,

Wiesbaden,

Germany)

were

coated

with

100

␮L

of

10

␮g/mL

OMVs

in

coating

buffer

(60

mM

carbonate

buffer,

pH

9.6).

Afterwards,

unspecific

binding

sites

were

saturated

with

3%

bovine

serum

albumin

(BSA)

in

PBS

for

1

h

at

RT.

Sera

from

mice

were

serially

diluted

in

PBS

with

1%

BSA

and

incubated

overnight

at

RT.

After

intense

washing

with

PBS

Tween

20

(PBS-T)

buffer,

the

alkaline

phosphatase

(AP)-conjugated

detection

antibody,

class-

specific

goat

anti-mouse

IgG/IgA

(Sigma)

for

sera,

was

added

for

1

h

at

37

◦

C.

The

detection

reaction

was

performed

by

incubating

the

sample

with

ABTS

substrate

for

20

min

at

room

temperature.

Absorbance

was

measured

with

an

ELISA

reader

(Sunrise

remote;

Tecan-Austria,

Groeding,

Austria)

at

a

wavelength

of

405

nm.

2.5.2.

Quantification

of

cytokines

from

sera

Cytokines

(IL-2,

IL-4,

IL-5,

IL-6,

IL-10,

IL-12(p40),

IL-12(p70),

IL-

13,

IL17,

IFN-

␥,

and

tumor

necrosis

factor)

were

quantified

from

serum

by

luminex-based

multiplex

assay

(Milliplex;

Millipore,

Bil-

lerica,

MA)

using

a

Bioplex

analyzer

(Bio-Rad,

Hercules,

CA).

2.6.

Statistics

Statistical

analyses

were

performed

using

GraphPad

Prism

5

for

Mac

OS

X.

All

experiments

were

performed

with

n

=

6.

Statis-

tical

comparisons

between

antibody

serum

levels

were

performed

using

Kruskal–Wallis

test,

followed

by

Dunn’s

post

hoc

test.

The

statistical

significance

was

set

at

P

<

0.05.

For

cytokine

levels,

it

was

performed

using

single-factor

analysis

of

variance,

followed

by

Turkey’s

post

hoc

test.

The

statistical

significance

was

set

at

P

<

0.001.

The

Kaplan–Meyer

curves

were

used

for

analysis

of

the

protection

experiment.

3.

Results

3.1.

Isolation

and

characterization

of

S.

flexneri

OMVs

The

scanning

electron

microscopy

showed

that

the

OMVs

secreted

in

vitro

by

S.

flexneri

were

spherical,

with

an

average

diam-

eter

of

50

nm

(

Fig.

1

A).

The

yield

obtained

was

18

±

0,

04

␮g/mg

determined

after

lyophilisation

and

referred

to

the

original

cell

culture

dry

weight.

Quantitative

analysis

showed

that

protein

con-

tent

was

54.52

±

3.2%,

whereas

the

LPS

content

was

37.6

±

4.8%.

A

comparative

SDS-PAGE

analysis

of

the

OMVs

revealed

that

con-

tained

proteins

corresponded

to

the

OmpA,

34

kDa;

OmpC/OmpF,

38/42

kDa;

VirG,

120

kDa

(

Fig.

2

)

already

described

by

other

authors

as

the

main

inmunodominant

antigenic

proteins

[25,26]

.

As

expected,

the

outer

membrane

protein

enriched

fraction

and

the

purified

OMVs

showed

a

similar

profile.

Furthermore,

OMVs

con-

tained

bands

at

62

kDa,

42

kDa

and

38

kDa

that

correspond

with

IpaB,

IpaC

and

IpaD

respectively

(

Fig.

2

)

[27]

.

Immunoblot

assay

using

a

monoclonal

antibody

specific

to

IpaB

or

IpaC

demonstrated

that

these

proteins

were

located

on

vesicles

(

Fig.

2

),

confirming

the

observation

of

Kadurugamuwa

and

Beveridge

[28]

.

3.2.

Characterization

of

OMVs-containig

nanoparticles

The

yield

of

the

OMV

antigen-loaded

NPs

manufactured

in

rela-

tion

to

the

initial

amount

of

polymer

employed

was

consistent

(89%).

Vaccine

formulations

were

homogeneous

and

spherically

shaped

(

Fig.

1

A).

The

average

size

of

NP-OMV

was

197

nm

with

a

polydispersity

index

of

0.06.

The

Z

potential

of

NP

was

tested

before

and

after

OMV

encap-

sulation.

Results

suggest

that

OMV

is

at

least

partially

bound

on

the

NP

surface,

indicated

by

the

change

in

Z

of

NP.

Zeta

potential

of

A.I.

Camacho

et

al.

/

Vaccine

29 (2011) 8222–

8229

8225

Table

1

Immunization

protocol

and

administration

route

strategy.

OMV:

free

outer

membrane

vesicles

extract

from

Shigella

flexneri

2a.

NP-OMVs:

OMVs

loaded

nanoparticles

(PBS:

phosphate

buffered

saline).

OMVs

NP-OMVs

Intradermal

Dosage

(administered

1

×)

20

␮g

2.5

mg

NP-OMVs

(eqv.

20

␮g

OMVs)

Volume

(50

␮L

PBS/dose)

Nasal

Dosage

(administered

3

×,

8

h

interval)

3

␮g/nostril

416

␮g

NP-OMVs

(eqv.

3

␮g

OMVs)

Volume

(3

␮L

PBS/nostril)

Ocular

Dosage

(administered

3

×,

8

h

interval)

3

␮g/nostril

416

␮g

NP-OMVs

(eqv.

3

␮g

OMVs)

Volume

(3

␮L

PBS/eye)

Oral

Dosage

(administered

1

×)

100

␮g

12.5

mg

NP-OMVs

(eqv.

100

␮g

OMVs)

Volume

(200

␮L

PBS/dose)

Fig.

1.

(A)

Scanning

electron

micrograph

images

of

outer

membrane

vesicles

(OMVs)

from

Shigella

flexneri

2a

(up),

or

loaded

in

nanoparticles

(NP-OMVs)

(down).

Scale

bar

indicates

200

nm.

(B)

Integrity

and

antigenicity

of

the

outer

membrane

vesicles

components

antigenic

components

after

encapsulation

into

nanoparticles.

Panel

shows

the

immunoblotting

developed

with

a

pool

of

sera

from

rabbit

hyperimmunized

with

whole

cells

from

Shigella

flexneri:

lanes

correspond

with

the

following

samples:

(1)

free

OMVs

and

(2)

OMVs

released

from

OMV-loaded

NPs.

free

OMVs

was

−14.1

±

3

mV.

The

encapsulation

of

the

extract

in

nanoparticles

resulted

in

a

change

of

Z

potential

from

−44

±

4

mV

to

−27

±

4

mV

when

OMVs

were

loaded

into

PVM/MA

nanoparticles.

To

further

confirm

OMV

encapsulation

into

NPs,

BCA

pro-

tein

determination

and

SDS-PAGE/immunoblotting

were

also

Fig.

2.

Comparative

analysis

of

Shigella

flexneri

outer

membrane

vesicles.

SDS-PAGE

with

silver

staining

for

proteins

(A)

or

for

LPS

(B),

and

immunoblotting

(C)

of

(1)

outer

membrane

vesicles

(OMVs),

(2)

extract

enrich

in

outer

membrane

proteins

(OMPs),

and

(3)

extract

enrich

in

Ipa

proteins.

Immunoblots

were

developed

with

polyclonal

antibodies

from

a

patient

infected

with

S.

flexneri

(lane

a),

anti-IpaC

mAb

(lane

b)

and

anti-IpaB

mAb

(lane

c).

Molecular

weight

markers

and

identity

of

some

bands

are

indicated.

performed.

The

procedure

involved

the

use

of

a

purification

step

in

order

to

discard

unbound

OMV.

S.

flexneri

OMVs

were

efficiently

associated

with

PVM/MA

nanoparticles,

as

they

showed

a

loading

encapsulation

of

20

␮g

OMVs/mg

of

polymer.

Besides,

an

immunoblotting

was

carried

out

using

sera

from

rabbit

hyper-

immunized

with

S.

flexneri.

Results

indicate

that

entrapment

in

nanoparticles

did

not

alter

its

antigenic

properties

(

Fig.

1

B).

3.3.

Evaluation

of

the

immunogenicity

and

protection

conferred

by

OMVs

vaccine

Groups

of

6

mice

were

immunized

once

by

intradermal

or

mucosal

routes

with

OMVs

(20

␮g/mouse),

either

free

or

encap-

sulated

in

NPs.

A

control

group

of

non-immunized

mice

was

also

included.

All

animals

immunized

by

nasal

or

ocular

routes

remained

in

good

health,

exhibiting

no

respiratory

difficulties,

changes

in

body

temperature,

or

abnormal

behaviour.

Oral

immu-

nized

mice

showed

a

transient

abdominal

swelling

a

few

hours

after

immunization.

By

contrast,

mice

immunized

intradermally

exper-

imented

sweating

and

lethargy

during

2

days

post-immunization,

which

disappeared

thereafter.

Specific

IgG2a

and

IgG1

against

OMVs

antigens

were

deter-

mined

by

indirect-ELISA

at

days

0,

15

and

35

post-immunization

(

Fig.

3

).

Results

expressed

that

the

OMV

immunization

by

either

route

elicited

significant

levels

of

serum

IgG1

and

IgG2a

with

respect

control

mice

(

Fig.

3

).

Higher

levels

of

IgG

were

found

in

groups

immunized

intradermally.

Overall

the

levels

of

IgG2a

(Th1

response)

were

higher

than

that

those

of

IgG1

(Th2).

An

adjuvant

effect

after

encapsulation

was

observed

on

the

immunogenicity

8226

A.I.

Camacho

et

al.

/

Vaccine

29 (2011) 8222–

8229

Fig.

3.

Antibody

immune

response

induced

after

vaccination

of

BALB/c

mice.

Serum

IgG1,

IgG2a

and

IgA

titers

in

vaccinated

mice

(n

=

6/group)

with

either

free

extract

(OMVs)

or

loaded

in

nanoparticles

(NP-OMVs)

at

weeks

0,

2

and

5

after

immunization.

Broken

line

indicates

first

dilution

tested.

Data

are

mean

value

(*P

<

0.05

for

immunized

mice

vs.

control).

(global

specific

antibody

response)

especially

after

oral

immuniza-

tion.

There

were

not

found

significant

differences

in

the

mucosal

levels

of

the

IgA

elicited

after

intradermal

or

mucosal

deliveries.

Levels

of

serum

cytokines

were

determined

at

day

15

post-

immunization

(

Fig.

4

).

The

encapsulation

of

OMVs

in

NPs

induced

an

increase

in

the

level

of

IL-12

(p40)

and

a

decrease

of

IL-10

with

respect

to

the

free

form,

by

intradermal

or

oral

delivery.

In

con-

trast,

after

ocular

or

nasal

immunization,

the

inverse

switching

phenomenon

was

observed.

At

day

35

after

immunization,

mice

were

challenged

with

S.

flexneri

via

intranasal

route

and

monitored

for

survival

over

30

days

(n

=

6

mice/group)

(

Fig.

5

).

Nasal

or

ocular

immunizations

with

free

OMVs

provided

complete

protection.

Non-significant

differences

were

found

between

OMV

free

or

nano-

encapsulated

in

groups

immunized

by

nasal,

ocular

or

oral

route.

In

contrast,

the

intradermally

delivery

of

free

OMVs

was

not

protective,

while

the

encapsulated

extract

conferred

full

protec-

tion.

4.

Discussion

Currently,

live

vaccines

provide

better

protection

as

compared

to

the

inactivated

vaccines,

including

the

acellular

ones

[2,29]

.

However,

it

is

always

difficult

to

properly

calibrate

attenuation

to

achieve

the

minimum

of

toxicity

with

the

optimal

immuno-

genicity.

Besides,

the

use

of

live

Shigella

vaccines

is

questionable

since

this

pathogen

is

able

to

strongly

interfere

with

the

immune

response,

by

inducing

an

immunosuppressive

condition

that

favors

infective

process.

In

our

present

experimental

study,

we

support

the

use

of

mucosal

immunization

with

acellular

vaccines.

Results

A.I.

Camacho

et

al.

/

Vaccine

29 (2011) 8222–

8229

8227

Fig.

4.

Immune

response

induced

after

vaccination

of

BALB/c

mice.

Cytokines

serum

level

(IL-10,

IL-12

(p40),

IL-12

(p70),

IL-5,

and

IFN-

␥)

detected

at

day

15

after

immunization

with

either

free

outer

membrane

vesicles

(OMVs)

(gray

bars)

or

loaded

in

nanoparticles

(NP-OMVs)

(black

bars).

Broken

line

indicates

serum

level

before

immunization.

Data

are

mean

value

(*P

<

0.001).

demonstrated

a

significant

efficacy

and

no

reactogenicity

in

the

mice

pulmonary

model.

The

best

prophylactic

measure

probably

would

be

to

prevent

bacterial

invasion

by

neutralizing

key

surface

virulence

factors.

The

outer

membrane

(OM)

of

Shigella

contains

several

main

vir-

ulence

factors,

including

outer

membrane

proteins

(OMP),

protein

adhesins,

the

highly

conserved

virulence-plasmid-encoded

Ipa

pro-

teins

[28]

as

well

as

LPS.

These

are

essential

components

in

the

invasion

process,

and

can

alter

the

course

of

infection

and

the

host

responses,

and

therefore

their

neutralization

for

the

host

will

suc-

ceed

in

protective

immunity

[25,30–33]

.

Outer

membrane

vesicles

(OMVs)

consist

of

OM

and

solu-

ble

periplasmic

components

shed

from

Gram-negative

bacteria.

This

blebbing

process

is

considered

as

a

peculiar

bacterial

extra-

cellular

secretion

system

than

enable

bacterial

colonization

and

impairs

host

immune

response

[34]

.

Therefore,

it

is

plausible

to

think

on

Shigella

OMVs

as

ideal

candidates

for

an

acellular

vac-

cine.

The

capacity

of

OMV-based

vaccines

to

stimulate

a

protective

immune

response

has

already

been

exploited

against

several

bac-

terial

pathogens,

such

as

Brucella

ovis

[18]

,

S.

typhimurium

[35]

,

Flavobacterium

[36]

,

Porfiromonas

[37]

or

Neisseria

meningitides

B,

with

over

55

million

doses

administered

to

date

of

the

former

[38]

.

As

many

Gram-negative

bacteria,

Shigella

bleb

off

membrane

vesicles

during

normal

growth.

Kadurugamuwa

and

Beveridge

already

obtained

and

characterized

membrane

vesicles

from

S.

flexneri

[28]

.

In

order

to

obtain

this

material

massively,

we

devel-

oped

an

extraction

protocol

that

also

maximize

OMV

purity.

Vesicles

were

isolated

from

concentrated,

cell-free

culture

super-

natant

leading

to

an

appropriate

antigenic

profile

as

well

as

high

purity

grade.

Besides,

final

product

was

ultradiafiltered

in

order

to

avoid

interferences

in

the

encapsulation

process.

OMV

used

here

contain

key

alarm

signals

such

as

LPS,

OMPs

and

Ipa

recognized

by

the

innate

immune

system,

including

epthe-

lial

cells,

MALT

and

antigen

presenting

cells

[39,40]

,

and

therefore

have

the

capacity

to

either

enhance

bacterial

clearance

or

cause

host

tissue

damage

by

activating

an

inflammatory

response.

It

is

interesting

to

note

that

these

components

provide

a

prolonged

stimulation

of

the

inflammatory

response

that,

at

first

instance,

facilitates

bacterial

survival

in

the

tissues.

However,

this

fact

will

lead

to

the

bacteria

elimination

by

the

host

immune

system

[41]

.

In

fact,

our

results

indicate

that

a

single

dose

of

non-adjuvated

OMVs

delivered

by

mucosal

routes

is

able

to

protect

against

a

lethal

challenge

with

S.

flexneri.

Vaccines

that

stimulate

protec-

tive

mucosal

immune

responses

often

need

an

adjuvant

for

proper

delivery

and

presentation

to

the

mucosal

immune

tissues.

The

mechanisms

underlying

the

effectiveness

of

free

OMV

without

external

adjuvant

may

be

explained

by

the

nature

of

some

individ-

ual

components

contained

within

this

“proteoliposome”

or/and

by

the

biophysical

properties

of

these

vesicles

[42]

.

Besides,

Ipa

con-

taining

OMVs

may

contribute

to

its

adjuvanticity

by

their

ability

to

interact

with

host

cell

receptors

which

facilitate

OMVs

transcyto-

sis

across

mucosal

epithelial

barriers

[27]

.

On

the

other

hand,

the

amphipatic

properties

of

OMVs

may

facilitate

its

own

movement

through

mucosal

tissues,

enhancing

antigen

presentation

to

drive

a

protective

response.

In

this

study,

we

measured

the

levels

of

cytokines

in

OMVs

vac-

cinated

mice

2

weeks

after

the

immunization.

Then,

we

analyzed

their

association

with

the

challenge

outcome.

A

strong

association

Fig.

5.

Protection

study

against

Shigella

flexneri.

BALB/c

mice

(20

±

1

g)

were

immunized

with

20

␮g

of

outer

membrane

vesicles

either

free

(OMVs)

or

loaded

into

nanoparticles

of

PVM/MA

(NP-OMVs)

by

intradermal

(

),

nasal

(

),

ocular

(

)

or

oral

(

),

routes.

An

extra

group

was

included

as

non-immunized

control

(

×).

At

day

35

after

immunization,

all

groups

received

an

intranasal

lethal

challenge

of

10

7

UFC/mouse

of

Shigella

flexneri

2a

(clinical

isolate).

Graphs

indicate

the

percentage

of

mice

that

survived

the

infective

challenge

at

the

indicated

days

after

immunization

(*P

<

0.01,

Logrank

test).

8228

A.I.

Camacho

et

al.

/

Vaccine

29 (2011) 8222–

8229

between

the

ratio

of

IL-12p40/IL-10

and

protection

was

found.

Moreover,

low

levels

of

IFN-

␥

correlated

with

protection.

However,

conclusions

from

these

particular

data

must

be

taken

with

caution

since

cytokine

levels

were

measured

directly

from

serum.

At

this

point,

further

studies

are

being

carried

out

to

really

establish

a

correlation

of

these

parameters

and

protection.

After

oral

administration,

under

steady-state

conditions,

some

factors

released

by

enterocytes,

such

as

retinoic

acid,

thymic

stromal

lymphopoietin

and

TGF-

␤,

will

“condition”

non-activated

resident

DCs

to

elicit

a

Th2

or

regulatory

responses

[43]

.

However,

following

an

inflammatory

stimulus,

a

recruitment

of

DC

express-

ing

CX3CR1

to

the

mucosal

tissues

is

observed,

increasing

the

number

of

DC

extending

dendrites

into

intestinal

lumen.

Under

this

state

of

high

activation,

DC-expressing

massively

co-stimulatory

molecules,

present

the

antigenic

determinant

to

the

specific

T

naïve

cells

in

the

T

area

MALT.

The

substantial

distinctive

release

of

IL-12

from

those

DCs

will

also

contribute

to

the

further

differentiation

of

naïve

cells

to

Th1/Th2/Th17,

linked

to

an

inflammatory

response.

Actually,

our

results

would

support

it

since

OMVs

adjuvanted

into

NPs

induced

increasing

levels

of

IL-12

(p40)

and

decreasing

IL-10

with

respect

to

the

free

form,

either

by

intradermal

or

oral

delivery.

NPs

can

enhance

the

delivery

of

the

loaded

antigen

to

the

gut

lym-

phoid

cells

due

to

their

ability

to

be

captured

and

internalized

by

cells

of

the

gut-associated

lymphoid

tissue

(GALT),

and

to

induced

maturation

of

DCs

with

a

significant

upregulation

of

CD40,

CD80,

and

CD86

and

a

Th1

response

in

animal

models.

The

mechanisms

responsible

for

DC

maturation

may

be

related

to

TLR-NP

specific

interaction

[5]

.

On

the

other

hand,

the

encapsulation

of

OMVs

in

NPs

induced

an

increase

in

the

level

of

IL-10

and

a

decrease

of

IL-12

(p40)

with

respect

to

the

free

form,

by

ocular

or

nasal

routes,

which

is

char-

acteristic

of

mucosal

adjuvants

that

usually

stimulate

a

Th2

T-cell

response

[44–46]

,

characterized

by

increased

secretory

IgA,

high

proportions

of

antigen-specific

serum

IgG1,

and

the

stimulation

and

synthesis

of

IL-4,

IL-5,

and

IL-10.

The

specific

immune

mechanisms

that

mediate

resistance

to

Shigella

infection

have

not

been

clearly

defined

and

are

currently

being

debated.

Thus,

in

humans,

up

regulation

of

both

proinflam-

matory

and

anti-inflammatory

are

observed

during

the

first

stages

of

infection.

Later,

in

relation

with

the

convalescent

stage

of

shigel-

losis,

an

increase

in

IFN-

␥

is

observed.

Summing

up,

although

Th1

is

effective

to

control

infection,

a

Th2

response

may

be

also

as

effective

but

shorter-lasting.

Concerning

the

antibody

response

elicited

after

OMV

immu-

nization,

we

cannot

establish

a

relation

between

antibody

levels

and

protection.

Serum

and

mucosal

antibodies

to

LPS

and

the

Ipa

proteins

have

been

demonstrated

during

human

shigellosis

[47,48]

.

However,

it

has

not

been

established

the

role

of

these

antibodies

to

limit

the

spread

or

severity

of

the

infection.

The

apparent

inconsis-

tency

between

IgG

subclass

response

and

cytokine

profile

may

be

due

to

immune

cells

other

than

T

helper

cells.

The

ultimate

goal

for

vaccination

is

to

stimulate

long-lasting

protective

immunological

memory.

Toll-like

receptors

[49]

gener-

ally

promote

adaptive

immune

responses

indirectly

by

activating

innate

immune

cells.

It

has

been

recently

shown

that

the

use

of

multiple

TLR-agonists

carried

by

nanoparticles

influence

in

the

induction

of

long-term

memory

cells

[50]

.

Recent

studies

report

that

in

a

murine

model

of

acute

bacte-

rial

infection

with

S.

flexneri

the

T

cell

response

is

dominated

by

the

induction

of

long-term

memory

Shigella-specific

Th17

cells

that

contribute

to

mediate

protective

immunity

against

reinfection

[51]

.

Now,

new

research

shows

an

unexpected

direct

role

for

TLR2

signalling

in

T

cells

themselves,

promoting

the

differentiation

and

proliferation

of

T

helper

17

(T

H

17)

cells

[49]

.

Taking

into

account

these

data

and

together

with

previous

results

from

our

own

group

about

the

high

ability

of

PVM/MA

to

stimulate

TLR2

[5]

suggest

that

these

nanoparticles

are

good

adjuvant

candidate

for

further

investigation.

OMVs

are

safe

and

protective

in

mice,

therefore,

the

use

of

OMVs

adjuvanted

into

NP

to

trigger

mucosal

immunity

and

effectively

neutralize

Shigella

infection

open

the

door

to

safely

deals

with

vaccination,

especially

critical

when

young

children

are

the

primary

target.

Acknowledgments

This

research

was

financially

supported

by

Health

Department

of

“Gobierno

de

Navarra”

(28/2007),

“Instituto

de

Salud

Carlos

III”

(PS09/01083

and

PI070326),

from

Spain.

Ana

Camacho

was

also

financially

supported

by

“Instituto

de

Salud

Carlos

III”

(FI08/00432).

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